When compared to splenic dendritic cells (DC), freshly-isolated liver DC produce lower levels of IL-12, secrete comparatively high levels of IL-10, and are inferior stimulators of naive allogeneic T cells. We have shown that liver DC are hyporesponsive to stimulation via the Toll-like receptor (TLR)4 pathway and that nuclear factor (NF)KB phosphorylation,- a critical determinant of DC maturation, is impaired. Moreover, stimulation with LPS confers resistance to subsequent stimulation with other TLR ligands. We have postulated that constant exposure of DC to LPS within the liver microenvironment further attenuates TLR4 and TLR9 signaling, invoking a mechanism(s) that prevents chronic liver inflammation. This phenomenon, resembling 'endotoxin tolerance' may contribute to the altered function of liver DC and to the inherent tolerogenicity of hepatic allografts. Mechanisms underlying endotoxin tolerance described in macrophages include elevated expression of negative regulators of the TLR signaling cascade. However, mechanisms underlying endotoxin toleranceTn DC in general, and in liver DC, in particular, are poorly defined. Our novel Preliminary data show that the signaling adaptor fJNAX-activating p_rotein of 12KDa (DAP12), that mediates inhibition of TLR activation, is upregulated in liver DC, and that intrahepatic IL- 6/ signal transducer and activator of transcription (STATS) signaling inhibits liver DC maturation and function. Consequently, we hypothesize that LPS in the liver induces negative regulators of TLR signaling, resulting in 'endotoxin tolerance' and impaired T cell allostimulatory capacity. We further postulate that TGF(3 and IL-10, expressed in the liver, potentiate endotoxin tolerance through molecular 'crosstalk' between their respective signaling pathways (smad for TGFP, and STATS for IL-6 and IL-10) and the TLR signaling pathway. The tolerogenic phenotype attributed to liver DC may contribute to the induction/maintenance of liver transplant tolerance, in the face of continuous endotoxin stimulation. Our studies are designed to provide new mechanistic insight into the molecular regulation of liver DC function and its impact on alloreactive T cell responses, both in vitro and in vivo. We propose the following specific aims: AIM 1: To elucidate the contributions of inducible regulators of TLR signaling to the development of endotoxin tolerance in liver DC. AIM 2: To ascertain the contribution of specific anti (IL-10; TGFP)- and pro-inflammatory mediators (HMGB1) expressed in the liver in the steady state and during injury, to endotoxin tolerance in liver DC. AIM 3: To establish the contribution of donor DC, TLR4, and negative regulators of TLR signaling in liver DC to allogeneic T cell responses following experimental liver transplantation. AIM 4: To determine the maturation status of hepatic DC, including their expression of negative regulators of maturation, in relation to human liver transplant outcome. Relevance: Dendritic leukocytes are now recognized as critical regulators of innate and adaptive immunity. Elucidation of mechanisms whereby the function of these cells is regulated within the liver environment and following liver transplantation, may lead to improved strategies for improved long-term liver transplant outcome.